Microvesicles (MVs) of different sub-cellular origin, and released by most cells, have been recently indicated as a novel way of intercellular communication (Al-Nedawi et al., 2009; Cocucci et al., 2009; Ratajczak et al., 2006). MVs include microparticles shed from the plasma membrane (shed MPs), also called ectosomes, and exosomes, secreted as a result of multivesicular bodies exocytosis. Both organelles mediate the transfer of lipids, proteins and genetic material from donor to target cells. Because of their small size some MVs can move from the site of discharge by diffusion and represent important vectors of inflammatory or pathogenic agents (Ardoin, 2008; Doeuvre, 2009; Emmanouilidou; Vella, 2007)}. MVs of hematopoietic/endothelial origin and derived from brain tumors are emerging as new biomarkers of tissue damage. The authors have recently shown that microglia, upon in vitro activation, shed MVs containing pro-inflammatory cytokines (Bianco et al., 2005a; Bianco et al., 2005b).
Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. It is a chronic pathology, characterized, in 80-90% of cases, by an initial relapsing remitting course followed by a secondary progressive phase in which patients develop progressive neurological disorders. The pathological hallmark of MS is the presence within the CNS of inflammatory infiltrates, containing few autoreactive T cells, lymphocytes and microglia/macrophages (Prineas et al., 2001; Sriram and Rodriguez, 1997). MS brain is also characterized by elevated levels of the microglial-derived cytokine IL1β, which contributes to demyelination and progressive neuronal damage (Martin et al., 1995; Badovinac et al., 1998; Wiemann et al., 1998; Furlan et al., 1999; Hemmer et al., 2002; Pollak et al., 2003; Takahashi et al., 2003; Furlan et al., 2007).
The authors have recently shown that IL1β is contained in and released from microparticles (MPs) which are shed from the plasma membrane of microglia upon cell activation (Bianco et al., 2005; Bianco et al., 2009). Plasma membrane-derived MPs, together with exosomes—the latter secreted as a result of multivesicular bodies fusion with the plasma membrane—are extracellular microvesicles (MVs), involved in the intercellular transfer of lipid and protein components as well as of genetic materials from donor to target cells and are important vectors of inflammatory or pathogenic agents (Rajendran et al., 2006; Ratajczak et al., 2006; Vella et al., 2007; Simons and Raposo 2009; Bianco et al, 2009; Qu et al., 2009). Since microglia is known to be activated in all major steps of MS and elevated levels of IL1β are present in MS brain tissue, in the present invention it was verified whether the production of MVs from microglia, both MPs and exosomes, increases in the course of Experimental Autoimmune Encephalomyelitis (EAE), a rodent model of human MS, and contributes to neuronal damage. Indeed, although MVs deriving from blood cells, endothelial cells or tumors are emerging as new messengers/biomarkers unveiling an existing situation of damage—i.e. ischemic cerebrovascular accidents, transient ischemic attacks (Diamant et al. 2004; Morel et al. 2004b; Distler et al. 2005; Pilzer et al. 2005; Morel et al. 2006; Leroyer et al. 2008; Douvre et al. 2009; Huttner et al. 2008)—microglia-derived MVs have been only recently described and characterized in vitro (Bianco et al., 2005; Bianco et al., 2009). In particular, the possibility that these organelles may be present in vivo at concentrations varying in relation with the pathophysiological brain context has never been explored, as the few reports available on MVs in CNS pathologies have focused only on endothelial- or blood cells-derived MVs (Horstman et al., 2007).
For example, WO2009/100029 discloses a diagnostic method, for the detection of a disease or other medical conditions in a subject, by determining the concentration of microvesicles within a biological sample, in particular blood-derived urinary microvesicles.
The document WO2010/072410 describes a method for immobilizing microvesicles to a coated surface using a laminar flow as well as means and methods for detecting microvesicles and uses thereof for detecting or determining diseases related to microvesicles and related kit. In this document, microvesicles are isolated from platelet poor plasma.
There is therefore the need for a method that allows the diagnosis and/or prognosis of a neurological disorder characterized by an inflammatory process. The process and/or the disorder being preferably characterized by microglial activation. There is the need for a marker specific of microglial activation. Such marker, present in the CSF, can be used for diagnostic or prognostic purposes.
In the present invention, the authors investigated in great details whether the amount of myeloid MVs increase in vivo in the course of EAE, reflecting microglial activation and the development of the inflammatory plaque. Given that the cerebrospinal fluid (CSF) is in direct contact with brain cells, the authors tested the possibility that CSF may be a direct recipient of microglia shedding products. Quantitative analysis of microglia-derived MVs in the CSF of EAE mice indicated a strict association between levels of microglia-derived MVs and disease course. The present results identify microglial MVs as strong biomarkers for diagnosis and monitoring of MS, thus revealing that CSF has a greater potential than previously thought for diagnosis of neuro-inflammatory diseases.
The authors demonstrate in the present invention that the number of microglia/macrophages-derived MVs is significantly increased in the cerebrospinal fluid (CSF) of rodents and humans during diseases characterized by microglial activation. During multiple sclerosis, an autoimmune disease in which T cells reactive to myelin initiate an inflammatory response in the brain, MVs derived from microglia/macrophages deliver a pro-inflammatory signal to neighbouring cells and contribute to neuroinflammation. Thus, these findings demonstrate a previously unappreciated role for microglia/macrophage MVs in neuroinflammatory diseases and identify MVs as a novel biomarker of brain inflammation.
Whereas very few reports described the detection of platelet-, endothelial cell- and oligodendrocyte-derived MVs in the CSF during disease (Doeuvre et al., 2009; Scolding et al., 1989), the presence of MVs of microglia/macrophage origin has never been reported.